A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

ETH Zurich, Switzerland

Progress in microelectronics, sensors and optics is strongly dependent on the miniaturization of components, and the integration of nanoscale structures into applicable systems. In this regard, conventional top-down technologies such as lithography have limits concerning the dimensions and the choice of material. Therefore, several bottom-up approaches have been investigated to satisfy the need for structures with large aspect ratios in the nanometre regime. For further implementation, however, it is crucial to find methods to define position, orientation and length of the nanowires. In this study, we present a microchip to trap in situ formed bundles of nanowires in microsized cages and clamps, thereby enabling immobilisation, positioning and cutting-out of desired lengths. The microchip consists of two layers, one of which enables the formation of metal-organic nanowires at the interface of two co-flowing laminar streams. The other layer, separated by a thin and deflectable PDMS membrane, serves as the pneumatic control layer to impress microsized features ("donuts") onto the nanowires. In this way, a piece of the nanowire bundle with a prescribed length is immobilised inside the donut. Furthermore, partly open ring-shaped structures enabled trapping of hybrid wires and subsequent functionalisation with fluorescent beads. We believe that the method is a versatile approach to form and modify nanoscale structures via microscale tools, thereby enabling the construction of fully functional nanowire-based systems.

Controlling the length and location of in situ formed nanowires by means of microfluidic tools.

&#x5B83;&#x6700;&#x8FD1;&#x8868;&#x73B0;&#x51FA;&#x7684;&#x56FA;&#x6001;&#x7EB3;&#x7C73;&#x5C3A;&#x5EA6;&#x6BDB;&#x5B54; &#39;(&#x7EB3;&#x7C73;&#x5B54;) &#x53EF;&#x4EE5;&#x7528;&#x4F5C;&#x9AD8;&#x5EA6;&#x654F;&#x611F;&#x7684;&#x5355;&#x5206;&#x5B50;&#x4F20;&#x611F;&#x5668;&#x3002;&#x4F8B;&#x5982;&#xFF0C;&#x7535;&#x6CF3;&#x6613;&#x4F4D;&#x7684; DNA&#x3001; RNA &#x548C;&#x86CB;&#x767D;&#x8D28;&#x901A;&#x8FC7;&#x8FD9;&#x79CD;&#x7EB3;&#x7C73;&#x5B54;&#x5DF2;&#x542F;&#x7528;&#x68C0;&#x6D4B;&#x548C;&#x8FD9;&#x4E9B;&#x590D;&#x6742;&#x7684;&#x751F;&#x7269;&#x5206;&#x5B50;&#x7684;&#x7ED3;&#x6784;&#x5206;&#x6790;&#x3002;&#x7EB3;&#x7C73;&#x5B54;&#x5927;&#x5C0F;&#x63A7;&#x5236;&#x662F;&#x5173;&#x952E;&#xFF0C;&#x56E0;&#x4E3A;&#x6BDB;&#x5B54;&#x5FC5;&#x987B;&#x76F8;&#x5F53;&#x5927;&#x5C0F;&#x7684;&#x95EE;&#x9898;&#x7684;&#x5206;&#x6790;&#x7269;&#x5206;&#x5B50;&#x3002;&#x4F7F;&#x7528;&#x6700;&#x5E7F;&#x6CDB;&#x7684;&#x65B9;&#x6CD5;&#x57FA;&#x4E8E;&#x805A;&#x7126;&#x7535;&#x5B50;&#x6216;&#x79BB;&#x5B50;&#x5149;&#x675F;&#xFF0C;&#x56E0;&#x800C;&#x9700;&#x8981; &#xFF08;&#x626B;&#x63CF;&#xFF09; &#x900F;&#x5C04;&#x7535;&#x955C;&#x548C;&#x805A;&#x7126;&#x7684;&#x79BB;&#x5B50;&#x675F; (FIB) &#x4EEA;&#x5668;&#x4EEA;&#x8868;&#x7684;&#x5236;&#x9020;&#x3002;&#x5373;&#x4F7F;&#x5F88;&#x5C0F;&#x7684;&#x6BDB;&#x5B54;&#x5DF2;&#x7ECF;&#x4F7F;&#x7528;&#x8FD9;&#x4E9B;&#x65B9;&#x6CD5;&#xFF0C;&#x4ECD;&#x7136;&#x5B58;&#x5728;&#x8BB8;&#x591A;&#x95EE;&#x9898;&#x3002;&#x8FD9;&#x4E9B;&#x5305;&#x62EC;&#x88AB;&#x5C40;&#x9650;&#x4E8E;&#x6BD4;&#x8F83;&#x8584;&#xFF0C;&#x673A;&#x68B0;&#x8F83;&#x7A33;&#x5B9A;&#x819C;&#xFF0C;&#x7279;&#x522B;&#x662F;&#x5728;&#x4E2A;&#x4F4D;&#x6570;&#x7EB3;&#x7C73;&#x8303;&#x56F4;&#x5185;&#xFF0C;&#x5B54;&#x5F84;&#x7F3A;&#x4E4F;&#x5728;&#x53CA;&#x5185;&#x5B54;&#xFF0C;&#x8868;&#x9762;&#x7279;&#x6027;&#x7684;&#x63A7;&#x5236;&#x8981;&#x6C42;&#x548C;&#x6700;&#x540E;&#xFF0C;&#x5236;&#x9020;&#x6210;&#x672C;&#x3002;&#x5728;&#x6982;&#x5FF5;&#x9A8C;&#x8BC1;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x7684;&#x5C0F;&#x8BF4;&#x548C;&#x7B80;&#x5355;&#x8DEF;&#x7EBF;&#x5236;&#x5907;&#x91D1;&#x5C5E;&#x7EB3;&#x7C73;&#x5B54;&#x660E;&#x663E;&#x76F4;&#x5F84;&#x4F4E;&#x4E8E; 20 nm &#x5728;&#x89E3;&#x51B3;&#x65B9;&#x6848;&#x4E2D;&#x4F7F;&#x7528;&#x7535;&#x6C89;&#x79EF;&#x548C;&#x5B9E;&#x65F6;&#x79BB;&#x5B50;&#x7535;&#x6D41;&#x53CD;&#x9988;&#x3002;&#x8FD9;&#x79CD;&#x5236;&#x4F5C;&#x65B9;&#x6CD5;&#x63D2;&#x5165;&#x5230;&#x53EF;&#x4EE5;&#x4F7F;&#x7528;&#x7684;&#x5E73;&#x53F0;&#x79CD;&#x7C7B;&#x76F8;&#x5F53;&#x5927;&#x7684;&#x7075;&#x6D3B;&#x6027;&#x548C;&#x7EB3;&#x7C73;&#x5B54;&#x819C;&#x6750;&#x6599;&#x3002;&#x6211;&#x4EEC;&#x8981;&#x5927;&#x5F97;&#x591A;&#x7684;&#x6BDB;&#x5B54;&#xFF0C;&#x5F88;&#x7B80;&#x5355;&#xFF0C;&#x4F7F;&#x4F7F;&#x7528;&#x6C99;&#x51B0;&#x6216;&#x5176;&#x4ED6;&#x534A;&#x5BFC;&#x4F53;&#x5236;&#x9020;&#x65B9;&#x6CD5;&#xFF0C;&#x4ECE;&#x5F00;&#x59CB;&#x786B;&#x9178;&#x76D0;&#x7EB3;&#x7C73;&#x5B54;&#x63A5;&#x53E3;&#x5728; Pt &#x9540;&#x540C;&#x65F6;&#x76D1;&#x6D4B;&#x5176;&#x79BB;&#x5B50;&#x7535;&#x5BFC;&#x5728;&#x540C;&#x4E00;&#x65F6;&#x95F4;&#x5728;&#x53CC;&#x6052;&#x7535;&#x4F4D;&#x7684;&#x8BBE;&#x7F6E;&#x3002;&#x7531;&#x4E8E; Pt &#x7684;&#x6C89;&#x79EF;&#xFF0C;&#x7EB3;&#x7C73;&#x5B54;&#x8DCC;&#x5E45;&#x5927;&#x5C0F;&#xFF0C;&#x4ECE;&#x800C;&#x51CF;&#x5C11;&#x5B54;&#x9699;&#x7535;&#x5BFC;&#x3002;&#x4E00;&#x65E6;&#x8FBE;&#x5230;&#x6240;&#x9700;&#x7684;&#x5B54;&#x9699;&#x7535;&#x5BFC;&#x3001; &#x7535;&#x6C89;&#x79EF;&#x8FC7;&#x7A0B;&#x505C;&#x6B62;&#x901A;&#x8FC7;&#x5207;&#x6362;&#x819C;&#x7535;&#x6781;&#x7684;&#x6F5C;&#x529B;&#x548C;&#x5236;&#x4F5C;&#x8FC7;&#x7A0B;&#x5C31;&#x5B8C;&#x6210;&#x4E86;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x8868;&#x660E;&#x8FD9;&#x4E9B;&#x6BDB;&#x5B54;&#x53EF;&#x4EE5;&#x7528;&#x4E8E;&#x5355;&#x4E00;&#x751F;&#x7269;&#x5206;&#x5B50;&#x5206;&#x6790;&#xFF0C;&#x4F8B;&#x5982;&#xFF0C;&#x6211;&#x4EEC;&#x5728;&#x6982;&#x5FF5;&#x9A8C;&#x8BC1;&#x7814;&#x7A76;&#x4E2D;&#x4F7F;&#x7528;&#x7684; &lambda;-DNA&#x3002;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x505A;&#x6CD5;&#x662F;&#x9002;&#x7528;&#x4E8E;&#x5355;&#x4E2A;&#x7EB3;&#x7C73;&#x5B54;&#xFF0C;&#x4EE5;&#x53CA;&#x7EB3;&#x7C73;&#x5B54;&#x9635;&#x5217;&#xFF0C;&#x5E76;&#x53EF;&#x4EE5;&#x8F7B;&#x677E;&#x5730;&#x6269;&#x5C55;&#x5230;&#x975E;&#x94C2;&#x91D1;&#x5C5E;&#x77FF;&#x5E8A;&#x3002;

Sub 20 nm &#x56FA;&#x6001;&#x7EB3;&#x7C73;&#x5B54;&#x7684;&#x5355;&#x5206;&#x5B50; biosensing &#x7CBE;&#x786E;&#x7535;&#x5316;&#x5B66;&#x5236;&#x5907;&#x3002;

It has recently been shown that solid-state nanometer-scale pores ('nanopores') can be used as highly sensitive single-molecule sensors. For example, electrophoretic translocation of DNA, RNA and proteins through such nanopores has enabled both detection and structural analysis of these complex biomolecules. Control over the nanopore size is critical as the pore must be comparable in size to the analyte molecule in question. The most widely used fabrication methods are based on focused electron or ion beams and thus require (scanning) transmission electron microscopy and focused ion beam (FIB) instrumentation. Even though very small pores have been made using these approaches, several issues remain. These include the requirement of being restricted to rather thin, mechanically less stable membranes, particularly for pore diameters in the single-digit nanometer range, lack of control of the surface properties at and inside the nanopore, and finally, the fabrication cost. In the proof-of-concept study, we report on a novel and simple route for fabricating metal nanopores with apparent diameters below 20 nm using electrodeposition and real-time ionic current feedback in solution. This fabrication approach inserts considerable flexibility into the kinds of platforms that can be used and the nanopore membrane material. Starting from much larger pores, which are straightforward to make using FIB or other semiconductor fabrication methods, we electrodeposit Pt at the nanopore interface while monitoring its ionic conductance at the same time in a bi-potentiostatic setup. Due to the deposition of Pt, the nanopore decreases in size, resulting in a decrease of the pore conductance. Once a desired pore conductance has been reached, the electrodeposition process is stopped by switching the potential of the membrane electrode and the fabrication process is complete. Furthermore, we demonstrate that these pores can be used for single-biomolecule analysis, such as that of Î»-DNA, which we use in a proof-of-concept study. Importantly, our approach is applicable to single nanopores as well as nanopore arrays, and can easily be extended to deposits of metal other than Pt.

Precise electrochemical fabrication of sub-20 nm solid-state nanopores for single-molecule biosensing.

Il a r&eacute;cemment &eacute;t&eacute; d&eacute;montr&eacute; que les pores &agrave; l&#39;&eacute;tat solide &agrave; l&#39;&eacute;chelle du nanom&egrave;tre (&quot;nanopores&quot;) peuvent &ecirc;tre utilis&eacute;es comme hautement sensibles capteurs de mol&eacute;cules simples. Par exemple, translocation &eacute;lectrophor&eacute;tique de l&#39;ADN, l&#39;ARN et les prot&eacute;ines par le biais de ces nanopores a permis &agrave; d&eacute;tection et analyse structurale de ces biomol&eacute;cules complexes. Contr&ocirc;le sur la taille du nanopore est critique car le pore doit &ecirc;tre comparable en taille &agrave; la mol&eacute;cule de l&#39;analyte en question. Le plus couramment fabrication m&eacute;thodes reposent sur des faisceaux &eacute;lectrons ou ions focalis&eacute;s et n&eacute;cessitent donc une instrumentation (FIB) du faisceau ionique focalis&eacute; et microscopie &eacute;lectronique &agrave; transmission (balayage). Bien que tr&egrave;s petits pores ont &eacute;t&eacute; faites &agrave; l&#39;aide de ces approches, plusieurs questions restent. Il s&#39;agit de l&#39;exigence d&#39;&ecirc;tre restreint &agrave; plut&ocirc;t mince, m&eacute;caniquement moins membranes stables, en particulier pour les diam&egrave;tres de pores dans la gamme nanom&egrave;tre &agrave; un seul chiffre, manque de contr&ocirc;le des propri&eacute;t&eacute;s de la surface &agrave; et &agrave; l&#39;int&eacute;rieur de la nanopore, et enfin, la fabrication des co&ucirc;ts. Dans l&#39;&eacute;tude de validation, nous pr&eacute;sentons un roman et un itin&eacute;raire simple pour fabriquer des nanopores m&eacute;talliques d&#39;un diam&egrave;tre apparent en dessous de 20 nm &agrave; l&#39;aide d&#39;&eacute;lectrod&eacute;position et r&eacute;troaction en temps r&eacute;el de courante ionique en solution. Cette approche de fabrication ins&egrave;re une grande souplesse dans les types de plates-formes qui peuvent &ecirc;tre utilis&eacute;s et le mat&eacute;riel de membrane nanopore. &Agrave; partir de pores beaucoup plus gros, qui sont simples &agrave; faire &agrave; l&#39;aide de la FIB ou autres m&eacute;thodes de fabrication de semi-conducteurs, nous electrodeposit Pt &agrave; l&#39;interface nanopore tout en surveillant sa conductance ionique en m&ecirc;me temps dans une configuration bi-potentiostatique. En raison de la d&eacute;position du Pt, le nanopore diminue en taille, r&eacute;sultant en une diminution de la conductance de pore. Une fois une conductance de pore d&eacute;sir&eacute; est atteint, le processus d&#39;&eacute;lectrod&eacute;position est arr&ecirc;t&eacute; en activant le potentiel de l&#39;&eacute;lectrode &agrave; membrane et le processus de fabrication est termin&eacute;. En outre, nous montrons que ces pores peuvent &ecirc;tre utilis&eacute;s pour l&#39;analyse de single-biomolecule, comme celui de &lambda;-ADN, que nous utilisons dans une &eacute;tude de validation. Important, notre approche est applicable aux nanopores unique ainsi que les tableaux nanopore et peut facilement &ecirc;tre &eacute;tendu aux d&eacute;p&ocirc;ts de m&eacute;taux autres que les Pt.

Pr&eacute;cis fabrication &eacute;lectrochimique de sous-20 nm &agrave; semi-conducteurs nanopores de biod&eacute;tection de mol&eacute;cules simples.

Vor kurzem hat sich gezeigt, dass Solid-State-Nanometerbereich Poren (&#39;Nanopores&#39;) als hochsensible Einzelmolek&uuml;l-Sensoren verwendet werden k&ouml;nnen. Beispielsweise hat die elektrophoretische Translokation von DNA, RNA und Proteine durch solche Nanopores Erkennung und Strukturanalyse von diesen komplexen Biomolek&uuml;len erm&ouml;glicht. Kontrolle &uuml;ber die Nanopore Gr&ouml;&szlig;e ist entscheidend, da die Pore Analyten betreffenden Molek&uuml;ls in der Gr&ouml;&szlig;e vergleichbar sein muss. Die meistverbreitete Fabrikation Methoden basieren auf fokussierte Elektronen oder Ionen Balken und ben&ouml;tigen daher, dass (Scan) Transmissionselektronenmikroskopie und focused Ion beam (FIB) Instrumentierung. Obwohl sehr kleine Poren mit diesen Ans&auml;tzen vorgenommen wurden, bleiben einige Fragen. Dazu geh&ouml;ren die Voraussetzung des Seins beschr&auml;nkt auf eher d&uuml;nn, mechanisch weniger stabile Membranen, besonders f&uuml;r Pore Durchmesser im einstelligen Nanometerbereich, fehlende Kontrolle &uuml;ber die Oberfl&auml;cheneigenschaften auf und innerhalb der Nanopore, und schlie&szlig;lich die Herstellung Kosten. In der Proof-of-Concept-Studie, wir berichten &uuml;ber eine neuartige und einfache Route f&uuml;r die Herstellung von Metall-Nanopores mit scheinbaren Durchmessern unter 20 nm mit Electrodeposition und Ionischen aktuelle Feedback in Echtzeit in L&ouml;sung. Dieser Fabrikation-Ansatz f&uuml;gt betr&auml;chtliche Flexibilit&auml;t in der m&ouml;glichen Plattformen, die verwendet werden k&ouml;nnen und die Nanopore Membran-Material. Ausgehend vom viel gr&ouml;&szlig;eren Poren, die einfach zu machen, mit FIB oder andere Halbleiter-Fertigung-Methoden sind, electrodeposit wir Pt an der Nanopore-Schnittstelle w&auml;hrend der &Uuml;berwachung seiner Ionische Leitf&auml;higkeit zur gleichen Zeit in einem Bi-potentiostatische-Setup. Durch die Ablagerung von Pt verringert sich die Nanopore in der Gr&ouml;&szlig;e, was zu einem R&uuml;ckgang von den Leitwert Pore. Sobald eine gew&uuml;nschte Pore Leitwert erreicht hat, der Electrodeposition-Prozess wird beendet, indem man das Potential der Membran-Elektrode und der Prozess abgeschlossen ist. Dar&uuml;ber hinaus zeigen wir, dass diese Poren f&uuml;r Einzel-Biomolek&uuml;l Analyse, wie die des &lambda;-DNA, verwendet werden k&ouml;nnen, die wir, in einem Proof-of-Concept-Studie verwenden. Wichtig ist, unseren Ansatz gilt f&uuml;r einzelne Nanopores sowie Nanopore-Arrays, und kann leicht erweitert werden, Ablagerungen von Metall als Pt.

Pr&auml;zise elektrochemische Herstellung Sub-20 nm-Solid-State-Nanopores f&uuml;r Einzelmolek&uuml;l-Biosensing.

Recentemente &egrave; stato dimostrato che i pori su scala nanometrica a stato solido (&#39;nanopori&#39;) possono essere utilizzati come sensori singola molecola altamente sensibili. Per esempio, traslocazione elettroforetica del DNA, RNA e proteine attraverso tali nanopori ha permesso sia di rilevazione e analisi strutturale di queste biomolecole complesse. Controllo sulle dimensioni del nanopore &egrave; fondamentale come il poro deve essere paragonabile nel formato alla molecola dell&#39;analita in questione. Il pi&ugrave; diffuso fabbricazione metodi sono basati su fasci di elettroni o ioni focalizzati e pertanto necessitano di microscopia elettronica della trasmissione (scansione) e ionico focalizzato fascio strumentazione (FIB). Anche se molto piccoli pori sono state fatte usando questi approcci, diversi problemi rimangono. Questi includono il requisito di essere limitato a piuttosto sottile, meccanicamente meno stabile delle membrane, specialmente per i diametri dei pori nel campo del nanometro singola cifra, mancanza di controllo delle propriet&agrave; superficiali presso e dentro il nanopore, e infine, costo di fabbricazione. Lo studio di proof-of-concept, segnaliamo su un romanzo e semplice percorso per fabbricare nanopori metallo con diametri apparenti inferiore a 20 nm mediante elettrodeposizione e feedback in tempo reale di corrente ionica in soluzione. Questo approccio di fabbricazione inserisce una notevole flessibilit&agrave; in tipi di piattaforme che possono essere utilizzati e il materiale della membrana nanopore. A partire da pori molto pi&ugrave; grandi, che sono semplici da effettuare utilizzando FIB o altri metodi di fabbricazione dei semiconduttori, abbiamo electrodeposit Pt all&#39;interfaccia nanopore mentre la conduttanza ionica allo stesso tempo in una configurazione bi-potenziostatica il monitoraggio. A causa della deposizione di Pt, il nanopore diminuisce in dimensione, causando una diminuzione della conduttanza dei pori. Una volta raggiunta una conduttanza poro desiderata, viene interrotto il processo di elettrodeposizione commutando il potenziale dell&#39;elettrodo della membrana e del processo di fabbricazione. Inoltre, ci dimostrano che questi pori possono essere utilizzati per l&#39;analisi single-biomolecola, come quello di &lambda;-DNA, che usiamo in uno studio di proof-of-concept. Soprattutto, il nostro approccio &egrave; applicabile al singolo nanopori, nonch&eacute; nanopore matrici e pu&ograve; essere facilmente esteso ai depositi di metallo diverso da Pt.

Precisa fabbricazione elettrochimico di sub-20 nm a stato solido nanopori per singola molecola biosensori.

&#x6700;&#x8FD1;&#x3001;&#x30CA;&#x30CE;&#x30E1;&#x30FC;&#x30BF; &#x30B9;&#x30B1;&#x30FC;&#x30EB;&#x306E;&#x56FA;&#x4F53;&#x6BDB;&#x7A74; (&#39;&#x7D30;&#x5B54;&#39;&#xFF09; &#x9AD8;&#x611F;&#x5EA6;&#x5206;&#x5B50;&#x30BB;&#x30F3;&#x30B5;&#x30FC;&#x3068;&#x3057;&#x3066;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3068;&#x3048;&#x3070;&#x3001;DNA&#x3001;RNA &#x304A;&#x3088;&#x3073;&#x86CB;&#x767D;&#x8CEA;&#x306A;&#x3069;&#x306E;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x3092;&#x901A;&#x3057;&#x3066;&#x306E;&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x306E;&#x8EE2;&#x6D41;&#x691C;&#x51FA;&#x3068;&#x3053;&#x308C;&#x3089;&#x8907;&#x96D1;&#x306A;&#x751F;&#x4F53;&#x5206;&#x5B50;&#x306E;&#x69CB;&#x9020;&#x89E3;&#x6790;&#x306E;&#x4E21;&#x65B9;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x30B5;&#x30A4;&#x30BA;&#x3092;&#x5236;&#x5FA1;&#x306F;&#x3001;&#x7D30;&#x5B54;&#x3092;&#x4E00;&#x4F53;&#x554F;&#x984C;&#x306E;&#x5927;&#x304D;&#x3055;&#x306B;&#x5339;&#x6575;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3068;&#x3057;&#x3066;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;&#x6700;&#x3082;&#x5E83;&#x304F;&#x4F7F;&#x308F;&#x308C;&#x305F;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x96C6;&#x675F;&#x96FB;&#x5B50;&#x307E;&#x305F;&#x306F;&#x30A4;&#x30AA;&#x30F3; &#x30D3;&#x30FC;&#x30E0;&#x4E0A;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x304A;&#x308A;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066; (&#x30B9;&#x30AD;&#x30E3;&#x30F3;) &#x96FB;&#x5B50;&#x9855;&#x5FAE;&#x93E1;&#x304A;&#x3088;&#x3073;&#x96C6;&#x675F;&#x30A4;&#x30AA;&#x30F3;&#x30D3;&#x30FC;&#x30E0; (FIB) &#x8A08;&#x6E2C;&#x30D3;&#x30FC;&#x30E0;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3059;&#x308B;&#x4F5C;&#x88FD;&#x3002;&#x975E;&#x5E38;&#x306B;&#x5C0F;&#x3055;&#x306A;&#x5B54;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x884C;&#x308F;&#x308C;&#x3066;&#x3044;&#x308B;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x554F;&#x984C;&#x304C;&#x6B8B;&#x308B;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x8981;&#x4EF6;&#x8868;&#x9762;&#x306E;&#x6027;&#x8CEA;&#x3068;&#x3001;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x5185;&#x306E;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x3080;&#x3057;&#x308D;&#x8584;&#x3044;&#x6A5F;&#x68B0;&#x7684;&#x306B;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x819C;&#x3001;&#x5B54;&#x5F84; 1 &#x6841;&#x30CA;&#x30CE;&#x30E1;&#x30FC;&#x30C8;&#x30EB;&#x306E;&#x7BC4;&#x56F2;&#x3067;&#x306F;&#x7279;&#x306B;&#x4EE5;&#x4E0B;&#x306B;&#x5236;&#x9650;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x4E0D;&#x8DB3;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x306B;&#x306F;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3001;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x88FD;&#x9020;&#x30B3;&#x30B9;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x5C0F;&#x8AAC;&#x3068;&#x898B;&#x304B;&#x3051;&#x306E;&#x76F4;&#x5F84; 20 &#x4EE5;&#x4E0B;&#x3092;&#x6301;&#x3064;&#x91D1;&#x5C5E;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x3092;&#x88FD;&#x9020;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5358;&#x7D14;&#x306A;&#x30EB;&#x30FC;&#x30C8;&#x5831;&#x544A;&#x6982;&#x5FF5;&#x5B9F;&#x8A3C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;nm &#x306E;&#x96FB;&#x7740;&#x3068;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x306E;&#x30A4;&#x30AA;&#x30F3;&#x96FB;&#x6D41;&#x30D5;&#x30A3;&#x30FC;&#x30C9;&#x30D0;&#x30C3;&#x30AF; &#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x4F7F;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x4F5C;&#x88FD;&#x65B9;&#x6CD5;&#x306F;&#x975E;&#x5E38;&#x306B;&#x67D4;&#x8EDF;&#x306B;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306E;&#x7A2E;&#x985E;&#x304A;&#x3088;&#x3073;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x819C;&#x6750;&#x6599;&#x3092;&#x633F;&#x5165;&#x3057;&#x307E;&#x3059;&#x3002;FIB &#x305D;&#x306E;&#x4ED6;&#x534A;&#x5C0E;&#x4F53;&#x306E;&#x4F5C;&#x88FD;&#x65B9;&#x6CD5;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x306F;&#x7C21;&#x5358;&#x3067;&#x3059;&#x3001;&#x306F;&#x308B;&#x304B;&#x306B;&#x5927;&#x304D;&#x3044;&#x6BDB;&#x7A74;&#x304B;&#x3089;&#x59CB;&#x3081;&#x3066;&#x79C1;&#x305F;&#x3061; Pt &#x30CA;&#x30CE;&#x30DD;&#x30A2; &#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x3067;&#x305D;&#x306E; bi &#x30AA;&#x30FC;&#x30B9;&#x30C6;&#x30CA;&#x30A4;&#x30C8;&#x30B9;&#x30C6;&#x30F3;&#x30EC;&#x30B9; &#x30BB;&#x30C3;&#x30C8;&#x30A2;&#x30C3;&#x30D7;&#x3067;&#x540C;&#x6642;&#x306B;&#x30A4;&#x30AA;&#x30F3;&#x4F1D;&#x5C0E;&#x3092;&#x76E3;&#x8996;&#x3057;&#x306A;&#x304C;&#x3089; electrodeposit &#x3057;&#x307E;&#x3059;&#x3002;Pt &#x306E;&#x6C88;&#x6BBF;&#x306E;&#x305F;&#x3081;&#x3001;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x30B5;&#x30A4;&#x30BA;&#x3001;&#x6C17;&#x5B54;&#x30B3;&#x30F3;&#x30C0;&#x30AF;&#x30BF;&#x30F3;&#x30B9;&#x306E;&#x4F4E;&#x4E0B;&#x3067;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3059;&#x3002;&#x671B;&#x307E;&#x3057;&#x3044;&#x6C17;&#x5B54;&#x30B3;&#x30F3;&#x30C0;&#x30AF;&#x30BF;&#x30F3;&#x30B9;&#x306B;&#x9054;&#x3057;&#x305F;&#x819C;&#x96FB;&#x6975;&#x306E;&#x96FB;&#x4F4D;&#x3092;&#x5207;&#x308A;&#x66FF;&#x3048;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x96FB;&#x7740;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x304C;&#x505C;&#x6B62;&#x3057;&#x3001;&#x4F5C;&#x88FD;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x304C;&#x5B8C;&#x4E86;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x6BDB;&#x7A74;&#x306E;&#x6982;&#x5FF5;&#x5B9F;&#x8A3C;&#x7814;&#x7A76;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B; &lambda; DNA &#x306E;&#x3053;&#x3068;&#x306A;&#x3069;&#x306E;&#x5358;&#x4E00;&#x751F;&#x4F53;&#x5206;&#x5B50;&#x89E3;&#x6790;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x306F;&#x3001;&#x6211;&#x3005; &#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x3001;&#x5358;&#x4E00;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x3068;&#x3057;&#x3066;&#x30CA;&#x30CE;&#x7D30;&#x5B54;&#x30A2;&#x30EC;&#x30A4;&#x306B;&#x9069;&#x7528;&#x3055;&#x308C;&#x3001;Pt &#x4EE5;&#x5916;&#x306E;&#x91D1;&#x5C5E;&#x306E;&#x5806;&#x7A4D;&#x7269;&#x3092;&#x7C21;&#x5358;&#x306B;&#x62E1;&#x5F35;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

&#x30B5;&#x30D6; 20 nm &#x56FA;&#x4F53;&#x30CA;&#x30CE;&#x30DD;&#x30A2;&#x5358;&#x4E00;&#x5206;&#x5B50;&#x30D0;&#x30A4;&#x30AA;&#x30BB;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x7CBE;&#x5BC6;&#x306A;&#x96FB;&#x6C17;&#x5316;&#x5B66;&#x7684;&#x4F5C;&#x88FD;

&#xADF8;&#xAC83;&#xC740; &#xCD5C;&#xADFC;&#xC5D0; &#xB9E4;&#xC6B0; &#xBBFC;&#xAC10;&#xD55C; &#xB2E8;&#xC77C; &#xBD84;&#xC790; &#xC13C;&#xC11C;&#xB85C; &#xACE0;&#xCCB4; &#xB098;&#xB178;&#xBBF8;&#xD130; &#xC2A4;&#xCF00;&#xC77C;&#xC758; &#xC228; &#xAD6C;&#xBA4D; (&#39;nanopores&#39;)&#xB97C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC74C;&#xC744; &#xD45C;&#xC2DC; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC608;&#xB97C; &#xB4E4;&#xC5B4;, DNA, RNA&#xC640; &#xAC19;&#xC740; nanopores &#xD1B5;&#xD574; &#xB2E8;&#xBC31;&#xC9C8; &#xC804;&#xAE30; &#xC774;&#xB3D9; &#xC804; &#xAC80;&#xC0C9; &#xBC0F; &#xC774;&#xB7EC;&#xD55C; &#xBCF5;&#xC7A1; &#xD55C; biomolecules&#xC758; &#xAD6C;&#xC870; &#xBD84;&#xC11D; &#xC218; &#xC788;&#xAC8C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. Nanopore &#xD06C;&#xAE30; &#xC81C;&#xC5B4;&#xB294; &#xAE30; &#xACF5;&#xC5D0; &#xC2E4;&#xC815;&#xC774; &#xBD84;&#xC790; &#xD06C;&#xAE30; &#xBE44;&#xAD50; &#xD574;&#xC57C; &#xD558;&#xB294; &#xC911;&#xC694; &#xD55C;. &#xAC00;&#xC7A5; &#xC81C;&#xC870; &#xBC29;&#xBC95;&#xACFC; &#xCD08;&#xC810; &#xC744;&#xB41C; &#xC804;&#xC790; &#xB610;&#xB294; &#xC774;&#xC628; &#xBE54; &#xAE30;&#xBC18; (&#xC2A4;&#xCE94;) &#xC804;&#xC1A1; &#xC804;&#xC790; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; &#xBC0F; &#xC9D1;&#xC911;&#xB41C; &#xC774;&#xC628; &#xBE54; (FIB) &#xACC4;&#xCE21; &#xD544;&#xC694;&#xB85C; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xB110;&#xB9AC; &#xC0AC;&#xC6A9; &#xB418;&#xB294;. &#xC544;&#xC8FC; &#xC791;&#xC740; &#xC228; &#xAD6C;&#xBA4D; &#xC774;&#xB7EC;&#xD55C; &#xBC29;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB9CC;&#xB4E4;&#xC5B4;&#xC9C4;, &#xD558;&#xC9C0;&#xB9CC; &#xBA87; &#xAC00;&#xC9C0; &#xBB38;&#xC81C;&#xAC00; &#xB0A8;&#xC544; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xD3EC;&#xD568; &#xB418; &#xACE0; nanopore, &#xB0B4;&#xBD80; &#xD45C;&#xBA74; &#xD2B9;&#xC131; &#xC81C;&#xC5B4;&#xC758; &#xD2B9;&#xD788; &#xD55C; &#xC790;&#xB9AC; &#xB098;&#xB178;&#xBBF8;&#xD130; &#xBC94;&#xC704;&#xC758; &#xAE30; &#xACF5; &#xC9C1;&#xACBD;&#xC5D0; &#xB300; &#xD55C; &#xC548;&#xC815; &#xB9C9; &#xB35C; &#xAE30;&#xACC4;&#xC801;&#xC73C;&#xB85C; &#xC624;&#xD788;&#xB824; &#xC587;&#xC740; &#xC81C;&#xD55C; &#xBD80;&#xC871;&#xC758; &#xC694;&#xAD6C; &#xADF8;&#xB9AC;&#xACE0; &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC81C;&#xC870; &#xBE44;&#xC6A9;. &#xAC1C;&#xB150; &#xC99D;&#xBA85; &#xC5F0;&#xAD6C;&#xC5D0; &#xC6B0;&#xB9AC; &#xC18C;&#xC124;&#xACFC; &#xB0A0;&#xC870; 20 &#xC544;&#xB798; &#xBA85;&#xBC31;&#xD55C; &#xC9C1;&#xACBD;&#xC744; &#xAC00;&#xC9C4; &#xAE08;&#xC18D; nanopores&#xB97C; &#xC704;&#xD55C; &#xAC04;&#xB2E8;&#xD55C; &#xACBD;&#xB85C; &#xB300; &#xD55C; &#xBCF4;&#xACE0;&#xC11C; nm electrodeposition &#xBC0F; &#xC2E4;&#xC2DC;&#xAC04; &#xC774;&#xC628; &#xC804;&#xB958; &#xD53C;&#xB4DC;&#xBC31;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC194;&#xB8E8;&#xC158;&#xC5D0;&#xC11C;. &#xC774; &#xC81C;&#xC870; &#xBC29;&#xBC95;&#xC740; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xD50C;&#xB7AB;&#xD3FC;&#xC758; &#xC885;&#xB958;&#xC5D0; &#xC0C1;&#xB2F9;&#xD55C; &#xC720;&#xC5F0;&#xC131;&#xACFC; nanopore &#xBA64;&#xBE0C;&#xB808;&#xC778; &#xC18C;&#xC7AC;&#xB97C; &#xC0BD;&#xC785; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xB3D9;&#xC131; potentiostatic &#xC124;&#xCE58; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0;&#xC11C; &#xB3D9;&#xC2DC;&#xC5D0; &#xC774;&#xC628; &#xC804;&#xB3C4;&#xB3C4; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xB3D9;&#xC548; nanopore &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xC5D0; Pt electrodeposit &#xAC04;&#xB2E8; &#xAC70;&#xC9D3;&#xB9D0; &#xB610;&#xB294; &#xAE30;&#xD0C0; &#xBC18;&#xB3C4;&#xCCB4; &#xC81C;&#xC870; &#xBC29;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558;&#xB294;, &#xD6E8;&#xC52C; &#xB354; &#xD070; &#xC228; &#xAD6C;&#xBA4D;&#xC5D0;&#xC11C; &#xC2DC;&#xC791;. Pt &#xC99D; &#xCC29; &#xB54C;&#xBB38;&#xC5D0; nanopore &#xD06C;&#xAE30;, &#xAE30; &#xACF5; &#xC804;&#xB3C4;&#xB3C4;&#xC758; &#xAC10;&#xC18C;&#xC5D0; &#xB530;&#xB978; &#xAC10;&#xC18C; &#xD569;&#xB2C8;&#xB2E4;. &#xC6D0;&#xD558;&#xB294; &#xAE30; &#xACF5; &#xC804;&#xB3C4;&#xB3C4; &#xB3C4;&#xB2EC; &#xD558;&#xBA74; electrodeposition &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xB9C9; &#xC804;&#xADF9;&#xC758; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xC804;&#xD658; &#xD558; &#xC5EC; &#xC911;&#xC9C0; &#xD558; &#xACE0; &#xC81C;&#xC870; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xAC00; &#xC644;&#xB8CC; &#xB418; &#xBA74;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xB7EC;&#xD55C; &#xBAA8; &#xACF5; &lambda;-dna, &#xC6B0;&#xB9AC;&#xAC00; &#xAC1C;&#xB150; &#xC99D;&#xBA85; &#xC5F0;&#xAD6C;&#xC5D0; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xB4F1; &#xB2E8;&#xC77C; biomolecule &#xBD84;&#xC11D;&#xC744; &#xC704;&#xD574; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, &#xC6B0;&#xB9AC;&#xC758; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC740; &#xB2E8;&#xC77C; nanopores nanopore &#xBC30;&#xC5F4;&#xC5D0; &#xC801;&#xC6A9; &#xB418;&#xBA70; Pt &#xC774;&#xC678;&#xC758; &#xAE08;&#xC18D; &#xC608;&#xAE08; &#xD558;&#xB3C4;&#xB85D; &#xC27D;&#xAC8C; &#xD655;&#xC7A5;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB2E8;&#xC77C; &#xBD84;&#xC790; biosensing&#xC5D0; &#xB300; &#xD55C; &#xD558;&#xC704;-20 nm &#xC194;&#xB9AC;&#xB4DC; &#xC2A4;&#xD14C;&#xC774;&#xD2B8; nanopores&#xC758; &#xC815;&#xD655;&#xD55C; &#xC804;&#xAE30; &#xD654;&#xD559; &#xC81C;&#xC870;.

Recientemente se ha demostrado que los poros de estado s&oacute;lido escala nanom&eacute;trica (&#39;interconectivos&#39;) pueden utilizarse como sensores de una sola mol&eacute;cula altamente sensibles. Por ejemplo, desplazamiento electrofor&eacute;tico de DNA, RNA y prote&iacute;nas a trav&eacute;s de tal interconectivos ha permitido tanto la detecci&oacute;n y an&aacute;lisis estructural de estas biomol&eacute;culas complejas. Control sobre el tama&ntilde;o de nanopore es fundamental ya que el poro debe ser comparable en tama&ntilde;o a la mol&eacute;cula de analito en cuesti&oacute;n. El m&aacute;s ampliamente utilizado fabricaci&oacute;n m&eacute;todos se basan en haces de electrones o iones enfocadas y por lo tanto requieren de microscopia electr&oacute;nica de la transmisi&oacute;n (exploraci&oacute;n) y ion enfocada la viga de instrumentaci&oacute;n (FIB). A pesar de poros muy peque&ntilde;os se han efectuado utilizando estos m&eacute;todos, siguen siendo cuestiones varias. Estos incluyen el requisito de ser restringida a algo fino, mec&aacute;nicamente menos estables membranas, especialmente para di&aacute;metros de poro en el rango del d&iacute;gito nan&oacute;metros, falta de control de las propiedades superficiales en y dentro de la nanopore, y finalmente, la fabricaci&oacute;n de costos. En el estudio proof-of-concept, Divulgamos sobre una novedosa y sencilla ruta para la fabricaci&oacute;n de metal interconectivos con aparentes di&aacute;metros por debajo de 20 nm mediante electrodeposici&oacute;n y retroalimentaci&oacute;n en tiempo real de corriente i&oacute;nica en soluci&oacute;n. Este enfoque de fabricaci&oacute;n inserta una considerable flexibilidad en los tipos de plataformas que pueden utilizarse y el material de la membrana nanopore. A partir de los poros mucho m&aacute;s grandes, que son sencillos hacer uso de FIB u otros m&eacute;todos de fabricaci&oacute;n de semiconductores, nos electrodeposit Pt en la interfaz de nanopore vigilando su conductancia i&oacute;nica al mismo tiempo en una configuraci&oacute;n de bi-potentiostatic. Debido a la deposici&oacute;n del Pt, el nanopore disminuye en tama&ntilde;o, resultando en una disminuci&oacute;n de la conductancia del poro. Una vez alcanzada una conductancia de poro deseada, se detiene el proceso de electrodeposici&oacute;n cambiando el potencial del electrodo de la membrana y el proceso de fabricaci&oacute;n. Adem&aacute;s, demostramos que estos poros pueden utilizarse para el an&aacute;lisis de la solo-biomol&eacute;culas, como el de &lambda;-DNA, que utilizamos en un estudio de prueba de concepto. Lo importante, nuestro enfoque es aplicable a interconectivos solo as&iacute; como matrices de nanopore y puede ampliarse f&aacute;cilmente a dep&oacute;sitos de metal que no sean de Pt.

Precisa fabricaci&oacute;n electroqu&iacute;mico de sub-20 nm interconectivos estado s&oacute;lido para una sola mol&eacute;cula de biosensores.

Full spectroscopic imaging by means of tip-enhanced Raman spectroscopy (TERS) was used to measure the distribution of two isomeric thiols (2-mercaptopyridine (2-PySH) and 4-mercaptopyridine (4-PySH)) in a self-assembled monolayer (SAM) on a gold surface. From a patterned sample created by microcontact printing, an image with full spectral information in every pixel was acquired. The spectroscopic data is in good agreement with the expected molecular distribution on the sample surface due to the microcontact printing process. Using specific marker bands at 1000 cm(-1) for 2-PySH and 1100 cm(-1) for 4-PySH, both isomers could be localized on the surface and semi-quantitative information was deduced from the band intensities. Even though nanometer size resolution information was not required, the large signal enhancement of TERS was employed here to detect a monolayer coverage of weakly scattering analytes that were not detectable with normal Raman spectroscopy, emphasizing the usefulness of TERS.

Tip-enhanced Raman spectroscopic imaging of patterned thiol monolayers.

For many drugs including antibiotics such as tetracyclines it is crucial that the molecule has the ability to quickly and passively permeate lipid membranes. Hence, the understanding of the permeability in relation to the molecular structure is an important aspect to rationally design novel pharmaceutically active compounds with high bioavailability. Here, we present a versatile method to study the kinetics of tetracycline permeation across liposome membranes on a microchip. Liposomes are immobilized onto the glass surface in a stripe pattern via an avidin-biotin bond and covered by microchannels to allow continuous delivery of tetracycline and buffer. The fluid flow provides a constant concentration profile and thereby resembles the drug transport via blood in the human body. Total internal reflection fluorescence (TIRF) microscopy was used to image the formation of a fluorescent drug-europium complex inside the liposomes. The permeation rates of various tetracyclines were investigated and the results compared to a conventional method (water-octanol partitioning). The findings largely confirm the correlation between membrane permeability and lipophilicity of the permeating molecules (Overton's rule). However, slight deviations reveal that lipophilicity is an important but not the exclusive parameter for the prediction of permeation. The method is fast enough to study the permeation of unstable tetracyclines such as rolitetracycline. Additionally, with the use of different cholesterol concentrations, the influence of membrane composition on the permeation rate can be investigated conveniently. The microfluidic approach can be easily applied to investigate the kinetics of other processes such as ligand-membrane receptor association and dissociation, provided that the process can be visualized by means of fluorescence spectroscopy.

A microfluidic vesicle screening platform: monitoring the lipid membrane permeability of tetracyclines.

We present a microfluidic device that enables the determination of intracellular biomolecules in multiple single cells. The cells are individually trapped and isolated in a microchamber array. Since the microchambers can be opened and closed reversibly, the cells can be exposed to different solutions sequentially, e.g. for incubation, washing steps, labelling and finally, for lysis. The tightly sealed microchambers enable the retention and analysis of cell lysate derived from single cells. The performance of the device is demonstrated by monitoring the levels of the cofactors NADPH and NADH both in healthy mammalian cells and in cells exposed to oxidative stress. The platform was also used to determine the toxic impact of the alkaloid camptothecin on the intracellular enzyme glucose-6-phosphate dehydrogenase levels. In general, the device is applicable for the analysis of cell auto-stimulation and the detection of intracellular metabolite concentration or expression levels of proteins.

A microchamber array for single cell isolation and analysis of intracellular biomolecules.

Immobilisation of liposomes and cells is often a prerequisite for long-term observations. The most common immobilisation approaches rely on surface modifications, encapsulation in porous materials or trapping in microfluidic channels by means of hurdle-like structures. While these approaches are useful for larger mammalian cells, the immobilisation of smaller organisms like bacteria and yeast or membrane model systems such as liposomes typically requires modification of their outer membrane to ensure that they are stably arrested at a defined position. Here, we present a protocol to immobilise biological objects, which can interact with hydrophobic cholesterol. A water-soluble molecule (cholesterol-PEG-biotin) is used as a linker, which can bind via avidin to biotinylated BSA (bBSA) previously absorbed on a glass surface. For better visualization, bBSA is arranged in a dot pattern by means of microcontact printing, and a microfluidic channel is used for sample supply. We show that our approach can be used to successfully immobilise artificial liposomes of different sizes, native (cell-derived) vesicles, vaccinia virions, Saccharomyces cerevisiae and Escherichia coli, simply by flushing the objects through the channel. Under these conditions, small liposomes and biological objects are stably arrested at high flow rates, while larger cells and liposomes can be released again by application of high shear stress. This protocol can be applied for long-term studies where fluids must be changed repeatedly, for measuring fast kinetics where rapid fluid exchange is essential, and to study the effects of shear stress.

A facile protocol for the immobilisation of vesicles, virus particles, bacteria, and yeast cells.

A microfluidic chip is presented for lysis and one-step RNA purification from bacteria. Bacteria are lysed by joule-heating followed by a gel electrophoresis step for clean-up and subsequent elution of small RNA. Bubble formation during electrophoresis at constant current is suppressed through the use of a silver chloride cathode and a silver anode. To prevent silver chloride sediment in the bulk solution, the anode was immersed in a saturated chloride solution. Salt bridges in the form of polyacrylamide gels are used that could be precisely patterned with the help of phaseguides. Bubble-free actuation could be performed for more than 20 min under a constant current. For longer actuation times, cathodic silver-chloride became depleted and a silver-chloride sediment formed in the anodic microchamber at increasing distance from the anode with time. The chip functioning was verified by extraction of transfer-messenger RNA from Escherichia coli and subsequent amplification using reverse transcription real-time PCR. Incorporation of salt bridges enables effective bubble free actuation of Ag/AgCl electrodes in a microfluidic chip. This opens up new possibilities in a surge towards fully integrated diagnostic cartridges that are miniaturized and disposable.

Bubble-free electrode actuation for micro-preparative scale electrophoresis of RNA.

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.

Phillip Kuhn

Department of Chemistry and Applied Biosciences

ETH Zurich, Switzerland

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Phillip Kuhn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry and Applied Biosciences

ETH Zurich, Switzerland

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Department of Chemistry and Applied Biosciences